It is often necessary to improve the solubility of a drug in physiological media in order to achieve effective clinical performance of injectable formulations of the drug. Peptide drugs are often poorly soluble in physiological media due to the presence of hydrophobic substituents.
Solubility problems can also lead to poor absorption by other routes of administration and in some cases suitable solubilising agents can aid the absorption of the drug by other routes, for example oral or nasal.
Exemplary peptide drugs that are so poorly soluble in physiological media are LHRH analogues and growth hormone releasing factor (GRF) peptides.
Luteinising hormone releasing hormone (LHRH or GNRH) is a decapeptide secreted by hypothalamus and capable of inducing the release of both LH and FSH. It has the following formula: pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH.sub.2 (SEQ ID No:1).
LHRH can either stimulate pituitary gonadotropin secretion or be a potent inhibitor. When administered in a precise pulsatile pattern LHRH can restore the normal cyclic gonadotropin secretion. Pulsatile administration of LHRH using a computerized pump was used with good results in the induction of ovulation in anovulatory women with hypothalamic dysfunction. When administered chronically, LHRH or its agonists proved to be potent inhibitors of gonadotropic secretion, providing a temporary (fully reversible) gonadotropin specific medical hypophisectomy.
To date, thousands of LHRH analogs have been synthesized, that can act either as agonists or antagonists. In order to produce LHRH antagonists, which work by receptor occupancy, it is necessary to substitute several amino acids on the LHRH molecule. Antagonists also require precise topological features to achieve high binding affinity to the receptor. There are a lot of LHRH analogues recently synthesized, in which the amino acids contain aromatic or other functional groups capable of the so-called hydrotropic interaction. The use of LHRH antagonists with their immediate inhibition of gonadotrophin release may be useful in therapeutic areas, such as contraception and in treatment of hormone-dependent disorders. In the case of hormone-dependent tumors, avoiding the initial stimulatory phase produced by LHRH agonists may be a particular advantage. For a review on LHRH analogues, see Karten and Rivier, 1986.
Antide, in particular, is a potent LHRH antagonist, with formula, biological activity and preparation as described in EP Patent 377,665.
From studies carried out by the Applicant, it resulted, for example, that antide has a very poor solubility in 0.9% NaCl solution (solubility 25 .mu.g/ml) or other isotonic media such as phosphate buffered saline (solubility was 16 .mu.g/ml). Previous aqueous formulations of antide have shown poor bioavailability and pharmacokinetic reproducibility. This is due to antide being present at the site of injection in concentrations above 25 .mu.g/ml for example, which leads to the formation of a precipitate on contact with the physiological medium. This precipitate can be gelatinous in nature and has a detrimental effect on drug absorption, as shown by clinical investigations carried out by the Applicant.
Other gonadotrophin releasing hormone antagonists in aqueous solutions can form gel structures and in addition, the solubility is known to increase as the pH of the solution is reduced, due to increased ionisation of the molecule (Cannon J. B, et al., 1995).
GRF (also called Somatorelin) is a peptide secreted by the hypothalamus which promotes the release of growth hormone from the anterior pituitary. It occurs naturally as 44-, 40-, and 37-amino acid peptides; the 44-amino acids form may possibly be converted into smaller forms, but all are reported to be active, the activity residing in the first 29 amino acid residues. A peptide corresponding to the 1-29 amino acid sequence of human GRF [hGRF(1-29)], also called Sermorelin, has been prepared by recombinant DNA technology as described in European Patent EP 105 759.
Sermorelin has been used in the form of acetate for the diagnosis and treatment of growth hormone deficiency.
GRF has a therapeutic value for the treatment of certain growth-hormone related disorders. The use of GRF to stimulate the release of GH is a physiologic method of inducing long bone growth or protein anabolism.
It is well known that the natural form of GRF can suffer from chemical degradation in aqueous solution, primarily of Asn at position 8 which results in reduced biological potency (Friedman et al., 1991; Bongers et al., 1992).
The main hydrolytic reactions occurring in GRF are sensitive to pH and reported to be: rearrangement of Asp.sup.3, at pH 4-6.5, cleavage of the Asp.sup.3 -Ala.sup.4 bond at pH 2.5-4.5, deamidation and rearrangement of Asn.sup.8 at pH above 7 (Felix A. M., 1991). Due to the combined degradation pathways, unstabilised aqueous solutions GRF are most stable in the pH range 4-5. Bongers et al. (Bongers et al., 1992) have shown that the deamidation reaction at Asn.sup.8 increases rapidly as the pH is raised above pH 3.
Various workers have made analogues of GRF by substitution of amino acids into the natural GRF sequence to improve the chemical stability (Serono Symposia USA, 1996; Friedman, 1991). While modification can be an effective means to improve the stability and retain bioactivity, it may be undesirable due to altered immunogenicity, which could be a problem for chronic therapies such as growth hormone deficiency.
It is known from the literature that, in certain cases, the addition of aromatic agents to solutions of proteins can cause a negative effect on solubility, resulting in precipitation. For example, when aromatic agents were brought into contact with recombinant human growth hormone (rhGH), conformational changes or denaturation occurred, resulting in the formation of rhGH aggregates. (Maa Y. F. and Hsu C. C., 1996). Additionally, to show that this was not a general phenomenon, it was shown that aromatic amino acid derivatives improved the solubility and enhanced the absorption of growth hormone (Leone Bay A, et al., 1996).
Nicotinamide has been reported to solubilise conventional pharmaceutical compounds (i.e. non-peptides with molecular weight less than 1000 daltons) by a process of charge transfer complexation, also called hydrotropic solubilisation. This process may result from the interaction of aromatic groups in the solubilising agent and aromatic or other suitable functional groups on the drug molecule. For example see Rasool et al., 1991.
However, the Applicant has found, and the corresponding data are here reported in the experimental section, that other molecules, containing aromatic groups such as benzoate or salicylate, which could interact by a hydrotropic mechanism (Jain N. K. and Patel V. V., 1986), show only a minor improvement in the solubilisation of an LHRH analogue (antide) in saline solution.
European Patent Application 0 649 655 describes the solubilisation of a water insoluble anti-ulcer drug using nicotinamide in order to produce a useful injectable form. Many potential derivatives of the active moiety are claimed, however, no in-vivo data were presented to demonstrate improved efficacy.
PCT Application WO 96/10417 describes the co-administration of Asp.sup.B28 human insulin and nicotinamide in order to achieve a rapid onset of action of the hypoglycaemic effect. The claimed nicotinamide concentration range was 0.01 to 1 M (0.1-12% w/w), but preferably from 0.05 to 0.5 M. The document gives evidence for faster absorption during an in-vivo study in pigs, however, a mechanism by which the improved absorption occurs is not addressed and, therefore, no generalisable teachings can be drawn from this document.